WO2002079782A1 - Reactif et procede pour l'immunoanalyse de l'elastase 1, et procede de detection de pathologie pancreatique - Google Patents

Reactif et procede pour l'immunoanalyse de l'elastase 1, et procede de detection de pathologie pancreatique Download PDF

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Publication number
WO2002079782A1
WO2002079782A1 PCT/JP2002/003255 JP0203255W WO02079782A1 WO 2002079782 A1 WO2002079782 A1 WO 2002079782A1 JP 0203255 W JP0203255 W JP 0203255W WO 02079782 A1 WO02079782 A1 WO 02079782A1
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Prior art keywords
elastase
latex
immunoassay
present
reagent
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PCT/JP2002/003255
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English (en)
Japanese (ja)
Inventor
Tokio Sawai
Katsuya Ohde
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Mitsubishi Kagaku Iatron, Inc.
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Priority to US10/473,387 priority Critical patent/US20040091940A1/en
Priority to JP2002577561A priority patent/JPWO2002079782A1/ja
Priority to EP02707280A priority patent/EP1385001A4/fr
Publication of WO2002079782A1 publication Critical patent/WO2002079782A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)

Definitions

  • the present invention relates to a reagent for immunoassay of elastase 1, an immunoassay method, and a method for detecting a disease.
  • analysis in this specification includes both “measurement” for quantitatively or semi-quantitatively determining the amount of an analyte and “detection” for determining the presence or absence of an analyte. Is included. Background branch art
  • Elastase includes elastase 1 and elastase 2.
  • the former elastase 1 is present in the blood vessels, and 90% of it is bound to ⁇ , 1-antitribosine in the blood, and elastase 1 is more clinically useful for measuring blood levels.
  • Elastase is also present in leukocytes, platelets, spleen, etc., and is distinguished immunologically from granulocyte elastase and ⁇ elastase, which provide information such as acute inflammation.
  • Rout elastase is an exocrine enzyme that specifically hydrolyzes elastin in connective tissue in blood.It is localized as proelastase in the acinar cells of Rout, and is secreted by the intestine and activated by tribcine. Is done. Elastase 1 shows abnormally high levels relatively early and frequently, reflecting inflammation associated with ⁇ cancer (especially ⁇ head), and is therefore useful as an index for the diagnosis or follow-up of rot disease. There are various tests for knee disease, and serum amylase has been measured for a long time. However, since amylase is also produced from organs other than ⁇ , it lacked specificity as a test for Rou disease, but elastase-1 has high specificity for ⁇ disease, so it must be measured accurately. Is particularly important in medical settings.
  • elastase 1 For the measurement of elastase 1, a method for measuring its enzymatic activity was developed, but its sensitivity and accuracy were insufficient.
  • immunological measurement for measuring elastase 1 as a protein amount is widely used, and RIA ( radioi mm unoassay).
  • elastase 1 in the sample and a certain amount of radioactive material Blood elastase 1 is analyzed by a so-called competitive method in which elastase 1 is competitively reacted with elastase 1 antibody and the radioactivity reacted with the antibody is measured.
  • RIA radioi mm unoassay
  • Elastase 1 shows a high level in rutoma, etc., but it is a spleen-specific enzyme, so it is also expected to be an indicator to monitor chronic rutitis instead of amylase. Nashi can be accurately grasped, the disclosure of c iNVENTION improvement of the prior art are important
  • An object of the present invention is to solve the above-mentioned drawbacks of the prior art, to perform analysis easily and without any special facilities and in a short time so as to be able to respond to an emergency test.
  • An object of the present invention is to provide an elastase 1 immunoassay reagent and an immunoassay method capable of quantitatively analyzing over a wide range from a concentration region to a high concentration region.
  • the present invention relates to a reagent for immunoassay for elastase 1, comprising two types of latex particles having different particle sizes and carrying two types of monoclonal antibodies having different specificities for elastase 1.
  • the present invention relates to a method of contacting latex particles having different particle diameters carrying two kinds of monoclonal antibodies having different specificities to elastase 1 with a test sample, and producing latex particles produced by an antigen-antibody reaction. Analyzing the degree of aggregation of particles, Elasta The present invention relates to a method for the immunoassay of ase1.
  • the present invention relates to a method for detecting a disease, wherein elastase 1 is analyzed by the immunoassay method.
  • FIG. 1 is a graph showing the correlation between the RIA method and the immunoassay method of the present invention.
  • FIG. 2 is a graph showing the minimum detection limit value in the immunoassay method of the present invention.
  • FIG. 3 is a graph showing the effective sensitivity in the immunoassay method of the present invention.
  • FIG. 4 is a graph showing the dilution linearity in the concentration range where the theoretical value is 0 to 600 ng ZdL in the immunoassay method of the present invention.
  • FIG. 5 is a graph showing the dilution linearity in the concentration range where the theoretical value is 0 to 600 ng / dL in the immunoassay method of the present invention.
  • the reagent for immunoassay of the present invention comprises two kinds of latex particles having different particle sizes, each carrying two kinds of monoclonal antibodies having different specificities for elastase 1, and preferably (1) a first monoclonal antibody for elastase 1 A first latex particle carrying an antibody, and (2) a second latex particle carrying a second monoclonal antibody to elastase 1 having a different specificity from the first monoclonal antibody and having a different particle size from the first latex particle. Latex particles.
  • the latex particles used in the present invention are not particularly limited as long as they are latex particles that can be used for ordinary immunoassay reagents.
  • examples include polystyrene and styrene-styrene sulfonic acid copolymer. be able to.
  • the average particle size of the latex particles can be appropriately selected depending on the detection concentration of the object to be measured or the measuring instrument, and can be, for example, 0.05 to 0.5. In the present invention, by using latex particles having different particle diameters, particularly from low to high values can be accurately analyzed.
  • the average particle size of the latex particles in the present invention means a value measured by an electron microscope.
  • the latex particles having a small particle diameter for example, particles having a particle diameter of 0.05 to 0.3 m are preferably used, and particles having a particle diameter of 0.05 to 0.25 ⁇ m are more preferably used.
  • the large-diameter latex particles for example, particles having a diameter of 0.2 to 0.5 m are preferably used, and particles having a diameter of 0.25 to 0.5 m are more preferably used.
  • the anti-elastase 1 antibody to be carried on the latex two kinds of monoclonal antibodies having different reaction specificities, that is, two kinds of monoclonal antibodies which recognize different epitopes are used.
  • first monoclonal antibody of the present invention one monoclonal antibody of the present invention and another monoclonal antibody used in combination therewith (hereinafter “the first monoclonal antibody of the present invention”).
  • the second monoclonal antibody is sometimes referred to as “the second monoclonal antibody.”
  • the distance between the two epitopes is such that they do not cause steric hindrance when used as a latex reagent. Needs to be
  • two types of monoclonal antibodies and two types of latex particles having different particle sizes are used in appropriate combination.
  • Two types of latex particles with different particle sizes Two types of monoclonal antibodies can be carried on each child, or the first monoclonal antibody of the present invention can be used as one of two types of latex particles having different particle sizes (hereinafter, referred to as first latex particles). ), And the second monoclonal antibody of the present invention can be carried on another latex particle (hereinafter, sometimes referred to as a second latex particle).
  • the most suitable conditions can be set in consideration of the size of the latex particles to be used, the ratio thereof, and / or the characteristics (for example, reaction rate) of the monoclonal antibody.
  • the mixing ratio of the latex particles having a large particle diameter and the latex particles having a small particle diameter is not particularly limited, but, for example, may be appropriately selected within a range of 10: 1 to 1:10. Can be.
  • the latex carrying the monoclonal antibody can be prepared by a known method, for example, sensitization by physical or chemical bonding.
  • the antibody may be an antibody fragment, for example, Fab 'or F (ab') 2 , in addition to the immunoglobulin molecule itself, and F (ab ') 2 is preferably used.
  • Fab 'or F (ab') 2 in addition to the immunoglobulin molecule itself, and F (ab ') 2 is preferably used.
  • a jump value (extremely higher than the actual amount of elastase 1) may be observed in some patient samples. Yes, by using F (ab ') 2 , the jump value phenomenon can be suppressed.
  • the immunoassay reagent of the present invention includes various additives that can be added to the latex reagent, in addition to the monoclonal antibody-carrying latex particles, for example, a buffer solution, an aggregation promoter (for example, a water-soluble polymer such as polyethylene glycol). , A non-specific reaction inhibitor (for example, an alkali metal salt or a saccharide), or a protein [for example, serum albumin (BSA)].
  • a buffer solution for example, an aggregation promoter (for example, a water-soluble polymer such as polyethylene glycol).
  • a non-specific reaction inhibitor for example, an alkali metal salt or a saccharide
  • a protein for example, serum albumin (BSA)].
  • the buffer a buffer having a buffering ability at a pH of 6 to 8.5 is preferable.
  • the buffer having a pH of 6 to 8.5 is a conventionally well-known buffer, and examples thereof include a Tris buffer, a phosphate buffer, and a good buffer.
  • the concentration of Tris in the Tris buffer is such that a latex agglutination reaction occurs when the Tris buffer is used.
  • the concentration is not particularly limited as long as it can achieve the predetermined tris concentration described below, but it should be 0.1 to 0.5 mol / L. Is preferred.
  • the tris concentration in the system for performing the latex agglutination reaction is not particularly limited as long as it is a concentration capable of suppressing the self-aggregation reaction of the latex particles, and the coexisting salt, protein, and / or saccharide Can be appropriately selected depending on the concentration of the additive such as.
  • the tris concentration in the system for performing the latex agglutination reaction is preferably 0.1 to 0.5 mol / L, more preferably 0.2 to 0.3 mol / L. If it is less than 0.1 moI, the latex particles may cause self-aggregation reaction.If it exceeds 0.5 moI / L, the antigen-antibody reaction is suppressed, and the detection sensitivity is reduced. May worsen.
  • the pH of the buffer is preferably 6 to 8.5. If the pH is out of this range, the latex particles may self-aggregate, or may cause inconvenience in measurement accuracy.
  • the immunoassay reagent of the present invention contains a buffer having a pH of 6 to 8.5, each latex particle carrying an antibody, pH As long as the buffer of 6-8.5 and the test sample can be contacted, the state of each monoclonal antibody-supported latex particle and the pH of the buffer of 6-8.5 in the immunoassay reagent are particularly It is not limited. That is, in this case, the form of the immunoassay reagent of the present invention is not particularly limited. For example, it includes both monoclonal antibody-supported latex particles and a buffer solution having a pH of 6 to 8.5.
  • It can be a liquid-based reagent, or a two-part reagent consisting of a first reagent containing latex particles carrying each monoclonal antibody and a second reagent that is a buffer solution with a pH of 6 to 8.5. It can also be a system reagent.
  • each latex particle carrying an antibody is brought into contact with a test sample under the conditions of pH 6 to 8.5, whereby the reaction between the antibody and the antigen is carried out.
  • Elastase 1 in the test sample can be analyzed by causing the resulting latex agglutination reaction and analyzing the degree of agglutination.
  • a test sample to which the immunoassay method of the present invention can be applied includes elastase 1
  • the test sample is not particularly limited as long as it is a test sample that may be contained, and examples thereof include a biological sample, more specifically, serum, plasma, urine, urea, or body fluid. .
  • each monoclonal antibody-supported latex particle can be brought into contact with a buffer solution having a pH of 6 to 8.5 in advance, and then the mixture can be brought into contact with a test sample. Can be brought into contact with a buffer solution having a pH of 6 to 8.5 in advance, and the resulting mixture can be brought into contact with each monoclonal antibody-supported latex particle.
  • the conditions for the antigen-antibody reaction in the immunoassay method of the present invention can be the same as those used in ordinary immunological latex turbidimetric analysis methods.
  • the reaction is preferably carried out at a pH of 6 to 8.5.
  • the reaction temperature is preferably from 0 to 50 ° C, more preferably from 20 to 40 ° C.
  • the reaction time can be appropriately determined.
  • a general-purpose automatic analyzer can complete the measurement in 10 to 15 minutes.
  • the degree of aggregation generated by the antigen-antibody reaction can be analyzed by a known analysis method, for example, an optical analysis method.
  • Examples of the optical analysis method include a method of irradiating a reaction liquid with light to analyze scattered light or transmitted light, and more specifically, scattered light intensity, absorbance, or transmitted light intensity.
  • the analysis can be performed by using an optical instrument for measuring the temperature.
  • a preferred measurement wavelength is 300 to 800 nm.
  • the increase or decrease of the scattered light intensity, the absorbance, or the transmitted light intensity can be determined by selecting the size and / or concentration of the latex particles to be used and setting the reaction time according to a known method. It can be performed by measuring. Also, these methods can be used in combination.
  • serum or plasma is used as a test sample, and elastase 1 in the test sample is analyzed by the immunoassay method of the present invention.
  • elastase 1 in the test sample is analyzed by the immunoassay method of the present invention.
  • Teng disease especially acute inflammation
  • the amount of elastase-1 in serum or plasma is at least 400 ng "dL, it can be determined that the disease is a rumor disease (particularly acute spleenitis).
  • the time required to return to the normal value after the acute symptoms subside is shorter for amylase and lipase than for amylase and lipase, and one week for elastase 1 and as long as one month for elastase 1 (amylase or lipase is several days).
  • the utility of the present invention which can measure elastase 1 simply, easily, and with high sensitivity, is extremely high.
  • the method of the present invention can solve all of these problems, and has a special effect that elastase 1 can be accurately measured in a wide range of concentrations because of the latex method, and that the disease can be diagnosed accurately. is there.
  • the present invention has made it possible to accurately measure even lower elastase 1 levels, so that it was necessary to rely on the measurement of amylase, which was not said to be specific to ruto. Monitoring can be performed more accurately, and ⁇ disease can be widely determined. According to the present invention, it is possible to easily and quickly measure elastase 1 from a low value to a high value, which could not be accurately measured by the conventional technology, without performing extra operations such as dilution of the sample or increase in the amount of elastase 1. Now you can do it. This is immeasurably high for the field of clinical testing.
  • a reagent for measuring elastase 1 in a latex agglutination reaction system is the first time, and by accurately measuring elastase 1 by using a latex agglutination reagent, diagnosis of a rumor disease can be further improved. Be able to grasp accurately.
  • the one-component reagent that is, the second buffer composed of ⁇ 6 to 8.5
  • An immunological analysis reagent of the present invention comprising one reagent and a second reagent composed of the first latex particles carrying the first monoclonal antibody and the second latex particles carrying the second monoclonal antibody was prepared.
  • elastase 1 as an immunogen, and using a complex of elastase 1 and -antitribucin, and elastase 1 as antigens for screening, and using elastase 1 and mono-anti-tribsine in a conventional manner.
  • a hybridoma producing an antibody capable of recognizing this complex was selected.
  • an antibody capable of latex agglutination with only the complex of elastase 1 and anti-trypsin was selected from them, and F (ab ') 2 anti-elastase 1 for immunoassay reagent was selected.
  • Antibodies were prepared.
  • the spleen is aseptically removed from the mouse, and the mouse myeloma cells (myeloma cells) SP cultured in advance 2/0 -A g 1 4 to about 2 X 1 0 7 pieces in 1 X 1 0 eight of the spleen cells were added, was subjected to cell fusion with 4 00 0 presence 40% polyethylene glycol. 0.1 mL of each was dispensed into a 96-well cell culture medium and cultured for 2 weeks in a HAT medium, and then the antibody in the culture supernatant was assayed.
  • the antibody-producing hybridomas were cloned by the limiting dilution method, and eight clones of antibody-producing strains reacting with the complex of elastase 1 and, -antitribcine were obtained.
  • the complex of elastase 1 and ", 1-antitrypsin was prepared by mixing the respective solutions at 37 ° C for 3 hours and leaving them at 4 ° C for ⁇ .
  • mice Each High Priestess dormer 1 X 1 0 7 or obtained, pristane (2, 6, 1 0, 1 4 - Te tetramethyl pentadecane) 0. 5 m L of 1 0-1 2-week-old BALB / C mice The mice were grown intraperitoneally 14 to 20 days after intraperitoneal administration after intraperitoneal injection to obtain anti-elastase 1 antibodies, respectively. Each of these monoclonal antibodies is supported on polystyrene latex particles, and two types of anti-elastase 1 antibodies ⁇ 1 and ⁇ 4 are left as antibodies that undergo agglutination reaction only with the complex of elastase 1 and anti-trypsin. I chose.
  • a complex of elastase 1 and ", 1-antitribine can also be used.
  • the F (ab ') 2 fraction of the pile elastase 1 antibody E1 prepared in Example 1 (1) was subjected to a concentration of 0.2 mgZmL at a concentration of 0.01 mol I / tris buffer (pH 8. 1 mL of polystyrene latex (solid content: 10% by weight) having an average particle size of 0.2 yum was added to 9 mL of the solution dissolved in 0), and the mixture was stirred at room temperature for 60 minutes.
  • Tris buffer solution (pH 8.0) containing 0.5% by weight of serum albumin (BSA) was added to the solution, and the mixture was stirred at room temperature for 60 minutes.
  • BSA serum albumin
  • the F (ab,) 2 fraction of the anti-elastase 1 antibody E4 prepared in Example 1 (1) was used as the antibody, and polystyrene having an average particle diameter of 0.3 m was used as the polystyrene latex.
  • an anti-elastase 1 antibody (E4) sensitized latex solution was prepared.
  • the two types of latex solutions were mixed and diluted with Tris buffer so that the optical density (OD) at 700 nm was about 2, to obtain an anti-elastase-1 monoclonal antibody-sensitized latex solution. .
  • Comparative Example 1 Comparative ffl Preparation of immunoassay reagent
  • the anti-elastase 1 antibody (E 4) sensitized latex solution was prepared by repeating the preparation of the antibody 1 (E 1) sensitized latex solution.
  • a latex solution was used.
  • the Tris buffer used was the one prepared in Example 1 (3) above.
  • Example 1 Using the immunoassay reagent of the present invention prepared in Example 1 for 80 samples of human serum and plasma in which the elastase 1 content was measured in advance by the RIA method, the elastase 1 content was determined according to the procedure described below. The content was measured.
  • each human serum and plasma were mixed with the Tris buffer prepared in Example 1 (3).
  • 65 ⁇ I of the anti-elastase 1 monoclonal antibody-sensitized latex solution prepared in Example 1 (2) was added and stirred.
  • the absorbance at a wavelength of 570 nm was measured.
  • the change in absorbance during this time was taken as the change in absorbance (AAbs).
  • the measurement was performed using a Hitachi automatic analyzer model 7 ⁇ 70.
  • the RIA method was performed according to the following procedure. That is, to 100 L of each human serum and plasma, 100 mL of Iodide Rasterase 1 solution 100 and 100 L of the first antibody (anti-human erasease 100 egan antibody) were added at 37 ° C. After incubating for 3 hours, 500 L of a second antibody (anti-Egret IgG goat antibody) which specifically reacts with the first antibody was added and mixed in. Eccentric separation for 20 minutes at room temperature (1 500-2) After performing 200G) and removing the supernatant, the elastase 1 content was determined by measuring the radioactivity of the precipitate.
  • Figure 2 shows the average value (unit- ⁇ g / d) of 10 measurements of elastase 1 concentration in each dilution
  • Figure 2 shows a value obtained by adding and subtracting a value obtained by multiplying the standard value (SD) by 2.6 to the average value, that is, a value obtained by subtracting the standard value (SD) from the average value.
  • the horizontal bars located at the top, center, and bottom of each vertical bar shown in Figure 2 are ⁇ mean + (2.6XSD) '', ⁇ mean '', and ⁇ mean-one (2. 6 XSD)).
  • the minimum detection sensitivity is between the range of “average value (2.6 XSD)” and the range of “average value (2.6 XSD)” of the control solution not containing elastase 1. It does not overlap [that is, the value of "mean value-(2.6XSD)” is larger than the value of "mean value + (2.6XSD)” of the control solution. "It can be set from the minimum concentration. As shown in FIG. 2, the minimum detection sensitivity (lower limit of measurement) of the reagent for immunological analysis of the present invention prepared in Example 1 was found to be 30 ng / dL.
  • the elastase 1 concentration was measured for each real sample by the immunoassay method of the present invention.
  • Tables 1 to 3 show the results of measuring the elastase 1 concentration for each real sample by the EIA method according to the procedure shown in Comparative Example 2 described below.
  • the uppermost columns of "Example 1", “Comparative Example 1", and “Comparative Example 2" are the results using the immunoassay reagent of the present invention prepared in Example 1, respectively.
  • 4 shows the results obtained using the comparative immunoassay reagent prepared in Comparative Example 1 and the results measured by the EIA method.
  • results shown in Table 1 are the results of the actual samples with the elastase ⁇ concentration of about 90 ng / d.
  • results of Tables 2 and 3 show that the elastase 1 concentration was about 900 ⁇ g / d. ng / dL and about 2100 ng / dL of actual samples.
  • Human serum containing elastase 1 antigen at a concentration of 600 ng / dL was used as a standard elastase 1 antigen solution.
  • This standard elastase 1 antigen solution was diluted to various concentrations shown in Table 4, and the elastase 1 concentration was measured three times for each of the diluted concentrations. The measurement was performed based on the procedure described in the evaluation column (1), except that 5 ⁇ L of each dilution was used instead of 5 ⁇ L of human serum.
  • Table 4 shows the results.
  • Figures 4 and 5 show the correlation between the theoretical values and the measured values.
  • FIG. 4 shows the result when the theoretical value is 0 to 600 ⁇ g / d
  • FIG. 5 shows the result when the theoretical value is 0 to 600 ng / dL. From the results in Fig. 4, as a correlation equation, equation (2):
  • the immunoassay method of the present invention has excellent correlation with RIA, has a wider measurement range than RIA method, and has good reproducibility.
  • Table 4 Theoretical values Measured values vs. theoretical values
  • the reactivity (the degree of aggregation) of the immunoassay reagent of the present invention prepared in Example 1 with respect to elastase II in various states was evaluated.
  • Table 5 shows the results. The results shown in Table 5 are shown as relative values when the reactivity to the elastase 1 ⁇ and 1 antitribosine complex is set at 100%.
  • “PMS F” shown in Table 5 is phenylmethanesulfonyl fluoride.
  • the reagent for immunoassay of the present invention aggregated a complex of elastase 1 and ", 1-antitriscine, but did not aggregate sole (free) elastase 1.
  • the elastase 1 concentration of each actual sample used in the evaluation (2) was measured by the EIA method.
  • the EIA method was performed according to the following procedure. That is, 10 L of a sample was added to a 96-well plastic well on which F (ab ') 2 anti-elastase 1 antibody E1 was immobilized, and the mixture was reacted at 37 with a phosphate buffer for 2 hours. After washing, peroxidase-labeled F (ab,) 2 anti-elastase 1 antibody E4 was added and reacted at 37 ° C for 1 hour.
  • elastase 1 can be analyzed simply and in a short time (for example, for 10 minutes) without requiring a special facility such as RIA. can do. Further, according to the immunoassay reagent or immunoassay method of the present invention, elastase 1 can be quantitatively analyzed over a wide range from a low concentration region to a high concentration region.
  • a rubella disease (particularly, acute rubella) can be detected easily and in a short time, and it is possible to respond to an urgent test.
  • a rubella disease particularly, acute rubella
  • the present invention has been described according to the specific embodiments. However, modifications and improvements obvious to those skilled in the art are included in the scope of the present invention.

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Abstract

La présente invention concerne un réactif d'immunoanalyse de l'élastase 1 contenant deux types de particules de latex porteuses de deux anticorps monoclonaux, présentant des spécificités différentes par rapport à l'élastase 1, et dont les diamètres particulaires sont différents. L'invention concerne également un procédé d'immunoanalyse de l'élastase 1 impliquant de prendre un échantillon test et de le mettre en contact avec deux types de particules de latex porteuses de deux anticorps monoclonaux, présentant des spécificités différentes par rapport à l'élastase 1, et dont les diamètres particulaires sont différents, puis d'analyser l'étendu de l'agrégation des particules de latex imputable à la réaction entre antigène et anticorps. L'invention concerne enfin un procédé de détection de pathologie pancréatique impliquant l'analyse de l'élastase selon le procédé d'immunoanalyse de l'invention. L'invention permet ainsi une analyse commode et rapide (généralement, moins de 10 minutes) de l'élastase 1, sans recourir à du matériel spécial comme dans le cas du RIA.
PCT/JP2002/003255 2001-03-30 2002-04-01 Reactif et procede pour l'immunoanalyse de l'elastase 1, et procede de detection de pathologie pancreatique WO2002079782A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US10/473,387 US20040091940A1 (en) 2001-03-30 2002-04-01 Reagent and method for immunoanalysis of elastase 1 and method of detecting pancreatic disease
JP2002577561A JPWO2002079782A1 (ja) 2001-03-30 2002-04-01 エラスターゼ１の免疫分析用試薬及び免疫分析方法並びに膵疾患の検出方法
EP02707280A EP1385001A4 (fr) 2001-03-30 2002-04-01 Reactif et procede pour l'immunoanalyse de l'elastase 1, et procede de detection de pathologie pancreatique

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JP2004191332A (ja) * 2002-12-13 2004-07-08 Mitsubishi Kagaku Iatron Inc 免疫学的分析試薬及び免疫学的分析方法
JP2004325416A (ja) * 2003-04-28 2004-11-18 Sekisui Chem Co Ltd 測定試薬用担体粒子ラテックス及び測定試薬
JP2005106609A (ja) * 2003-09-30 2005-04-21 Wako Pure Chem Ind Ltd 免疫学的測定用試薬
JP2006329959A (ja) * 2005-05-30 2006-12-07 Sekisui Chem Co Ltd 測定試薬用担体粒子及び測定試薬
JP2006329958A (ja) * 2005-05-30 2006-12-07 Sekisui Chem Co Ltd 測定試薬用担体粒子及び測定試薬
WO2007114337A1 (fr) * 2006-03-31 2007-10-11 Nissui Pharmaceutical Co., Ltd., Kit de reactif pour la reaction d'agglutination immune et procede pour tester l'antigene
WO2009128209A1 (fr) * 2008-04-14 2009-10-22 パナソニック株式会社 Méthode de détection et système de détection
WO2009136541A1 (fr) * 2008-05-09 2009-11-12 アークレイ株式会社 Procédé de production de particule porteuse insoluble, particule porteuse insoluble, réactif de mesure, outil d'analyse d'échantillon et procédé turbidimétrique immunologique
JP2017134067A (ja) * 2016-01-22 2017-08-03 協和メデックス株式会社 可溶性インターロイキン−２受容体の測定方法及び測定用試薬
JP2019528438A (ja) * 2016-07-29 2019-10-10 ダイアザイム ラボラトリーズ， インコーポレイテッド ビタミンｄをアッセイするための方法および組成物
WO2023100910A1 (fr) * 2021-11-30 2023-06-08 株式会社Ｌｓｉメディエンス Procédé de mesure d'élastase 1 dans des matières fécales

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JP2004191332A (ja) * 2002-12-13 2004-07-08 Mitsubishi Kagaku Iatron Inc 免疫学的分析試薬及び免疫学的分析方法
JP2004325416A (ja) * 2003-04-28 2004-11-18 Sekisui Chem Co Ltd 測定試薬用担体粒子ラテックス及び測定試薬
JP2005106609A (ja) * 2003-09-30 2005-04-21 Wako Pure Chem Ind Ltd 免疫学的測定用試薬
JP2006329959A (ja) * 2005-05-30 2006-12-07 Sekisui Chem Co Ltd 測定試薬用担体粒子及び測定試薬
JP2006329958A (ja) * 2005-05-30 2006-12-07 Sekisui Chem Co Ltd 測定試薬用担体粒子及び測定試薬
WO2007114337A1 (fr) * 2006-03-31 2007-10-11 Nissui Pharmaceutical Co., Ltd., Kit de reactif pour la reaction d'agglutination immune et procede pour tester l'antigene
JPWO2007114337A1 (ja) * 2006-03-31 2009-08-20 日水製薬株式会社 免疫凝集反応試薬キット及び抗原の測定方法
JP5199067B2 (ja) * 2006-03-31 2013-05-15 日水製薬株式会社 免疫凝集反応試薬キット及び抗原の測定方法
WO2009128209A1 (fr) * 2008-04-14 2009-10-22 パナソニック株式会社 Méthode de détection et système de détection
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WO2009136541A1 (fr) * 2008-05-09 2009-11-12 アークレイ株式会社 Procédé de production de particule porteuse insoluble, particule porteuse insoluble, réactif de mesure, outil d'analyse d'échantillon et procédé turbidimétrique immunologique
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JP2017134067A (ja) * 2016-01-22 2017-08-03 協和メデックス株式会社 可溶性インターロイキン−２受容体の測定方法及び測定用試薬
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JP2019528438A (ja) * 2016-07-29 2019-10-10 ダイアザイム ラボラトリーズ， インコーポレイテッド ビタミンｄをアッセイするための方法および組成物
JP2021119355A (ja) * 2016-07-29 2021-08-12 ダイアザイム ラボラトリーズ， インコーポレイテッド ビタミンｄをアッセイするための方法および組成物
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JP7177767B2 (ja) 2016-07-29 2022-11-24 ダイアザイム ラボラトリーズ， インコーポレイテッド ビタミンｄをアッセイするための方法および組成物
WO2023100910A1 (fr) * 2021-11-30 2023-06-08 株式会社Ｌｓｉメディエンス Procédé de mesure d'élastase 1 dans des matières fécales

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US20040091940A1 (en) 2004-05-13
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